The mentioned amino acids are very useful. Aspartate has been used as a raw material of aspartame, and lysine, threonine, methionine and tryptophan have been used as amino acids for feeds, foods and medicines. Asparagine, isoleucine, glutamic acid, glutamine, leucine, valine, alanine, proline, phenylalanine and tyrosine have been used as amino acids for foods and medicines. Also, homoserine and O-succinylhomoserine can be used as a precursors of amino acids production.
According to the continuous increase of oil price, development of an alternative energy using a recycling materials in nature is getting attention. Among many candidates for alternative energy source, BioDiesel obtained from plant oil and Bioethanol produced by fermentation are the most attractive candidates. BioDiesel indicates fatty acid methyl ester or fatty acid ethyl ester synthesized by esterification of methanol produced using plant oil as a substrate in the presence of a catalyst. During the synthesis, the byproduct, glycerol, is necessarily generated by 10% of the total weight.
Glycerol (C3H8O3) is converted from glucose (C6H12O6) by 1 step reduction, which can provide improved reducing power during the metabolism of a microorganism. Many products produced by fermentation require reducing power in their metabolic pathways. Therefore, if the glycerol could be effectively used as a substrate, the yield and productivity of the desired fermentation product would be improved. Despite the expectation, the studies on glycerol have been limited to reuterin (Talarico et. al., Antimicrob. Agents Chemother., 32:1854-1858 (1988)), 2,3-butanediol (Biebl, et al., Appl Microbiol. Biotechnol. 50:24-29 (1998)), 1,3-propanediol (Menzel, et. al., Enzyme Microb. Technol., 20:82-86 (1997)), succinic acid (Korean Patent No. 10-0313134), itaconic acid (U.S. Pat. No. 5,457,040), 3-hydroxypropanaldehyde (Doleyres et al. Appl. Microbiol. Biotechnol. 68(4):467-474 (2005)) and propionic acid (Himmi et al., Appl. Microbiol. Biotechnol., 53: 435-440 (2000)). That is because the price of glycerol is higher than that of any other carbon sources used for the fermentation in this industry. Now, studies to produce glycerol by fermentation are undergoing (Wang et al., Biotechnol. Adv., 19(3):201-223 (2001)).
However, with the increase of BioDiesel production, glycerol production is also increasing, resulting in the rapid reduction of the price. Accordingly, there has been a report that 1,3-propandiol (Gonzalez-Pajuelo et al., J. Ind. Microbiol. Biotechnol. 31: 442-446, (2004)), hydrogen and ethanol (Ito et al., J. Biosci. Bioeng., 100(3): 260-265 (2005)) have been produced by using the byproduct of BioDiesel including glycerol. However, no reports have been made to produce the most representative fermentation product, amino acids, and other major metabolites by using glycerol.
Glycerol has been produced so far in the industry of soap, fatty acid, wax and surfactants. However, as mentioned above, the problem to effectively treat byproduct including glycerol according to increase of BioDiesel production may occur. In the meantime, the price of the purified glycerol is also expected to be dropped. Therefore, the production of useful fermentation products using glycerol might bring effects more than expected.
The cases of using glycerol in microorganisms have been reported in E. coli and Klebsiella pneumoniae. In E. coli, extracellular glycerol infiltrates in cells by using GlpF, one of aquaglyceroporin having permeability for water, glycerol and urea, without energy consumption (Heller et al., J. Bacteriol. 144:274-278, (1980)). The glycerol is converted into glycerol-3-phosphate by glycerol kinase, which is converted again into dihydroxyacetone phosphate (DHAP) by glycerol-3-phosphate dehydrogenase and then converted into glyceroaldehyde-3-phosphate (G-3-P) by triosephosphate isomerase (TpiA), followed by final metabolism. (Lin E C, Annu. Rev. Microbiol. 30:535-578, (1976)). In the case that glycerol kinase has no activity, glycerol is converted into dihydroxyacetone (DHA) by glycerol dehydrogenase (Gdh), which is converted again into dihydroxyacetone phosphate (DHAP) by glycerol kinase or dihydroxyacetone kinase (DHA kinase), followed by conversion again into glyceraldehydes-3-phosphate (G-3-P) before final metabolism (Paulsen et al., Microbiology, 146:2343-2344, (2000)). Such glycerol metabolic pathway is regulated by various factors. Particularly, when glycerol and glucose are together, the wild type E. coli uses glucose first, exclusively, and then uses glycerol (Lin, Annu. Rev. Microbiol. 30:535-578, (1976)).
Microorganisms of Corynebacterium genus are the ones that have been widely used in industrial fields. For example, Corynebacterium glutamicum has been used for the production of such amino acids as lysine and monosodium glutamate, and C. ammoniagenes has been widely used for the production of nucleic acid by fermentation industrially. It has reported that Corynebacteria can use various carbon sources such as glucose and raw sugar for fermentation. It has also been reported that Corynebacteria can use xylose by the introduction of a gene such as xylAB (Kawaguchi et al., Appl. Envion. Microbiol. 72(5): 3418-3428 (2006)). However, there are rare cases reporting that Corynebacteria use glycerol as a carbon source. The case of using glycerol in Corynebacterium glutamicum are also very low (Korean Patent Application No. 2006-057633).
Among the microorganisms of Corynebacterium genus, only 4 microorganisms were analyzed to identify their total genome sequences. And, a complete gene using glycerol was found only in Corynebacterium diphtheriae and such genes involved in glycerol consumption as GlpF was deficient in other three microorganisms, Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium jeikeium. The deficiency of the gene is the major obstacle for Corynebacteria to use glycerol efficiently.